Roland E. Lippoldt

The optical rotatory and viscometric properties of γ-poly-D-glutamate☆

Abstract The optical rotatory and viscometric properties of the γ-linked polypeptide, γ-poly- D -glutamate, are shown to be markedly dependent on the ionic state of the α-carboxyl side chain group. From the influence of various positively charged ions on the specific rotation of γ-poly- D -glutamate the relative order of their binding has been deduced: H + > Pb ++ > Cd ++ > Ba ++ > Ba ++ ⪢ guanidinium ∼ Na + . The binding of the heavy metals follows quite closely their affinity constants to the carboxylate group in simple aliphatic acids. Optical rotational and viscometric evidence is reported which indicates that the un-ionized polypeptide exists in a hydrogen-bonded hypercoiled form in water.

Structural characterization of labeled clathrin and coated vesicles

Clathrin (8 S) and coated vesicles have been covalently labeled by using the sulfhydryl-labeling fluorescent probe N-(1-anilinonaphthalene)maleimide. A large increase in energy transfer from Trp to anilinonaphthalene (AN) residues was observed in clathrin in the pH range approximately 6.5-6.0, where the rate of clathrin self-association increased rapidly. The change in energy transfer was indicative of a conformational rearrangement, which could be responsible for the initiation of the clathrin self-association reaction to form coat structure. The AN label was found in both the coat and membrane proteins after dissociation of coated vesicles at pH 8.5. The labeled coat and membrane proteins readily recombined to form coated vesicles after reducing the pH to 6.5, indicating that the labeling did not interfere with the ability of clathrin to self-associate and interact with uncoated vesicles to form coat structure. A comparison of the AN fluorescence with the Coomassie blue pattern after electrophoresis in sodium dodecyl sulfate-gels revealed that a 180,000-Da protein (clathrin) was mainly labeled in coated vesicles, while a 110,000-Da protein was also strongly labeled in uncoated vesicles. AN-labeled baskets and coated vesicles have been prepared. Trypsin digestion reduced the sedimentation rate of baskets from 150 S to 120 S and of coated vesicles from 200 S to 150 S. Gel electrophoresis of baskets and coated vesicles showed extensive conversion of clathrin (Mr 180,000) to a product of Mr approximately equal to 110,000, suggesting equivalent structural organization of the coat in coated vesicles as in baskets. In both cases, the peptide(s) released from the vesicles by digestion were essentially free of fluorescent label. In the case of the uncoated vesicles, tryptic digestion released most of the proteins remaining after coat removal.

Easily assembled digital data acquisition system for the analytical ultracentrifuge

A system for the acquisition of digital data from the analytical ultracentrifuge which uses a commercially available data acquisition board, a standard IBM compatible personal computer (PC), and an interface circuit has been developed. The system uses the signal from the standard Beckman scanner. Preliminary analysis and data reduction are performed at the PC within minutes of data acquisition using simple commercially available software, and final data fitting is performed with a mainframe computer. Procedures are described which allow approach to equilibrium to be followed and attainment of equilibrium to be demonstrated. Data density of approximately 200 points per millimeter column height (approximately 500 points per 100 microliters of sample) allows the use of short columns and hence short run times. Only 2 min are required to collect a complete scan, which is recorded in a format suitable for direct analysis by standard spreadsheet software. This allows multiple sequential scans to be quickly recorded at equilibrium and averaged to reduce noise prior to analysis. The combination of characteristics allows molecular weight determinations to be performed relatively quickly with only a few micrograms of protein. The system is inexpensive and easy to assemble given the centrifuge and a PC.

Molecular transitions of human thyroxine-binding globulin

Abstract The molecular transitions of thyroxine-binding globulin in guanidinium chloride solutions have been evaluated by circular dichroism, tryptophanyl fluorescence, and the polarization of tryptophanyl fluorescence at neutral pH. Below 2 m guanidinium chloride, thyroxine-binding globulin undergoes a time-dependent irreversible transition which involves a randomization of almost half of the α-helical residues. At higher guanidinium chloride concentrations (2 to 5 m ) thyroxine-binding globulin undergoes several further reversible molecular transitions. It appears that the molecular species obtained from serum is not the most stable form of this protein. The species present in 2 m guanidinium chloride, however, can be recovered from more highly unfolded forms of the protein.

Effects of several antimalarials and phenothiazine compounds on the formation of coat structure from clathrin.

We have evaluated the effects of two phenothiazine and several antimalarial drugs on the rates of polymerization of 8S clathrin molecules to 300S coat structures. Most of the drugs investigated have been shown in other studies to inhibit receptor-mediated endocytosis through the coated pit regions of plasma membranes. The two types of drugs were found to accelerate the polymerization rate without having much effect on the size distribution of the polymer species. The activities of the drugs appear to depend on the dibasic moiety and a large, hydrophobic aromatic ring in their structures.

Acid-induced conformational change in hog thyroglobulin

Abstract The molecular behavior of hog thyroglobulin in acid solutions has been examined by fluorescence, absorption, proton binding, and velocity centrifugation. The precipitation of thyroglobulin which normally occurs in the pH region near its isoelectric point was prevented by reducing the salt concentration to 0.01 m and the protein concentrations to the lowest levels needed for measurements. The rate of denaturation is very slow near pH 5.0 but increases rapidly with decreasing pH. The molecular properties of acid-denatured thyroglobulin, though still retaining some aspects of its native tertiary structure, are quite different from those of the native protein. In the molecular transition: (a) buried tryptophanyl residues become exposed, (b) the anomalous dissociation of certain groups becomes normalized, (c) subunits are formed, and (d) the molecular form of thyroglobulin and its subunits becomes partially unfolded.

The stability and transitions of tryptic-digested clathrin

The pH-dependence of dissociation of trypsin-digested baskets has been determined by light scattering and compared with that of undigested baskets. Essentially no difference was found between the two types of baskets. The molecular transitions of clathrin derived from digested baskets have been studied by fluorescence spectra and polarization measurements and compared with those of undigested baskets. The transitions in both forms of clathrin were very similar. It is clear, therefore, that removal of about 1/3 of the mass from the distal portions of the arms of the clathrin triskelion does not affect its structural transitions. The interactions between clathrin molecules in the basket structure and those within the molecule appear, therefore, to remain intact in the smaller clathrin chains remaining after tryptic digestion. The function of the distal portion of the clathrin chain still awaits elucidation.